Background: Video-assisted thoracoscopic esophagectomy has been one of the most preferable surgical treatments for early esophageal cancer. Some scholars suggested that the thoracic duct should be routinely ligated to reduce the incidence of postoperative chylothorax, while another group raised an objection. As a classic indicator of immune function, T lymphocyte subsets can be applied to assess the effects of prophylactic thoracic duct ligation during thoracoscopic esophagectomy.Methods: A total of 60 patients were recruited and randomized into thoracic duct ligation group and nonligation group. Venous blood was collected before and after video-assisted esophagectomy. The lymphocyte count and percentage, T lymphocyte subsets percentage were measured with fully automatic hemacytometer analyzer and flow cytometry. The difference between two groups was compared with t-test and the classified data were compared with Chi-square test.Results: No significant difference was observed in peripheral blood CD3+, CD3+CD4+, and CD3+CD8+ lymphocyte percentage between the two groups before operation (P > 0.05). The mean value of peripheral blood CD3+, CD3+CD4+ lymphocyte percentage in ligation group was obviously less than that of in nonligation group after operation (P < 0.05). The mean of CD3+CD8+ lymphocyte percentage in ligation group was obviously higher than that of in nonligation group after operation (P < 0.05).Conclusion: Ligation of thoracic duct during esophagectomy could lead to decreased percentage of T lymphocyte and CD4+ Tlymphocyte, especially after arch of azygos vein had been transected. The thoracic duct should be selectively ligated during esophagectomy.

For a long time, traditional thoracotomy surgery has been the most important treatment for early esophageal cancer, at the same time, there are many shortcomings in this surgery including the large wound and high risk.[1] With the advances of medical science, video-assisted esophagectomy has been rapidly developed, with the advantages of minimal invasion, low complication rate, and rapid recovery.[2],[3] However, chylothorax after esophagectomy has still been a rare but potentially dangerous complication, with an incidence rate of approximately 2.7%–3.8%.[4] Some scholars suggested that the thoracic duct should be routinely ligated to reduce the incidence of postoperative chylothorax.[5] However, others proposed that the occurrence of postoperative chylothorax could not be reduced with prophylactic ligation of the thoracic duct, in addition, there may be unfavorable effects on overall survival of resectable esophageal cancer patients.[6],[7] Due to the lack of sufficient clinical data, management of thoracic duct remained controversial.[8] T lymphocyte played an important role in cancer cells fighting, killing, and surveillance, which has generally applied to evaluate the state of cell-mediated immunity.[9],[10] In a global context, flow cytometry has become a standard method for testing T lymphocyte subsets, even with corresponding guideline.[11] Almost all the mature T cells observed in the peripheral blood were marked with CD3. The two most important major T cell subsets were CD4+ and CD8+. Most T lymphocytes that expressed CD8 proteins were cytotoxic, while T lymphocytes that expressed CD4 protein were known as helper T cells, which promoted the proliferation, maturation, and immunologic functions of other cell types.[12]

> Methods

Patients

All recruited patients were diagnosed as squamous cell carcinoma according endscopy biopsy. The invasive range was also evaluated with endoscopic ultrasonography. Patients those staged T1 without obvious lymph node enlargement were enrolled in this study. No distant metastases were discovered on routine examination such as computed tomography scan, color Doppler ultrasound, Emission Computed Tomography (ECT) scan, and so on. Exclusion criteria included patients with endocrine, metabolic, or hematological system diseases. The patients in other stages confirmed by postoperative pathology were also excluded from the study. The study has been approved by the Committee on Medical Ethics of Taian City Central Hospital and written informed consent was obtained from all patients.

Surgical procedure

To identify thoracic duct easily, 100–150 mL olive oil was drunk by the patient 12 h before operation. Video-assisted thoracoscopic esophagectomy and cervical gastroesophagostomy were performed by the same group of surgeons. To free esophagus and clear lymph node, arch of azygos vein usually needed to be ligated and transected. Behind middle-lower segment esophagus, the thoracic duct could be easily recognized and separated between the descending aorta and the azygos vein.[13] The thoracic duct remained as a single channel from the cisterna chyli to the level of the ninth vertebra.[14],[15] Hence, thoracic duct was separated and double-ligated thoracic duct at a level of 2 cm above the diaphragm.

Venous blood was drawn before operation and on the 1st day after operation. The peripheral blood lymphocyte count and the percentage were determined with the help of fully automatic hemacytometer analyzer.

Immunofluorescence staining

A volume of 2 mL EDTA anticoagulant blood sample and 2 mL of RBC lysing solution were added into a test tube. The red blood cells were fully cracked after oscillation of 2 min. After centrifuging for 5 min at 1000 rpm, the peripheral blood mononuclear cells could be obtained by discarding supernatant. Washing with 0.01 mol/L phosphate buffer solution (PBS), the cell concentration was adjusted to a concentration of 2 × 106/ml. 100 uL cells suspension and 10 uL CD4-FITC/CD8-PE/CD3-Percp mAb were added into the tube; then the mixture was gently oscillated and incubated at room temperature for 30 min in the dark. Washing with PBS twice after centrifuging at 1500 rpm for 5 min, then the supernatant was discarded, and the cell pellets were resuspended with 0.5 mL 1% paraformaldehyde.[16]

Flow cytometric analysis

First, the samples were stained with three-color reagents and then analyzed by flow cytometer with CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA). By forward scatter and side scatter monitoring, white blood cell (WBC) was displayed, and lymphocyte populations were gated according to their size and granularity. The nonspecific binding was determined with isotype control tube, and marker was set for distinguishing fluorescence negative and positive cell populations. A minimum of 2,000 lymphocytes were initially acquired. The number of CD3, CD3/CD4, and CD3/CD8 positive lymphocytes could be analyzed based on their Percp, FITC and PE fluorescence respectively.[17] By this flow cytometric analysis, the percentages of T lymphocyte, CD4+ T lymphocyte, and CD8+ T lymphocyte in total lymphocytes could be reported.

Statistical analysis

All analyses were performed with the computer-assisted SAS 8.0. Qualitative variables were presented with percentages and quantitative variables were expressed as mean ± standard deviation. Chi-square test was applied to compare differences between two groups. Two sample t-test was performed to analysis lymphocyte count and percentage, T lymphocyte subsets percentage between two groups. Paired t-test was also performed to analyze the values before and after operation. P < 0.05 was considered statistically significant.

> Results

A total of 64 patients were enrolled in this study, 4 patients were excluded, with the reason of failure in recognizing thoracic duct, blood transfusion during operation, postoperative chylothorax, T2 stage, respectively. At last, the remaining 60 patients were randomly classified into thoracic duct ligation group (n = 28) and nonligation group (n = 32). No anastomotic fistula was observed, and the mortality was zero. There was no significant difference in age, gender and stage, as well as the incidence of anastomosis fistula and chylothorax between the two groups (P > 0.05) [Table 1]

The thoracic duct was just behind middle-lower segment esophagus and parallel to its, between the aorta and the azygos vein. In all case, thoracic duct was successfully separated and double ligated. Total surgery time was about 190 min, with the blood loss of <100 mL [Figure 1]

Regardless of preoperative or postoperative, no significant difference was observed in lymphocyte count and percentage between two groups (P > 0.05). However, we can see that the percentage of lymphocyte in ligation group was higher than that of in nonligation group before operation; however, it was lower than that of in nonligation group after operation. No significant difference was observed in peripheral blood CD3+, CD3+CD4+, and CD3+CD8+ lymphocyte percentage between two groups before operation (P > 0.05). The mean value of peripheral blood CD3+, CD3+CD4+ lymphocyte percentage in ligation group was obviously less than that of in nonligation group after operation (P < 0.05). The mean value of CD3+CD8+ lymphocyte percentage in ligation group was obviously higher than that of in nonligation group after operation (P < 0.05) [Table 2]

Figure 1: Patients were in the left lateral position. After freeing esophagus, the thoracic duct can be easily recognized and separated between the descending aorta and the azygos vein above the diaphragm

The incidence of esophageal cancer was high in local people. Through screening endoscopy examination, a lot of patients can be diagnosed at early stage. More than 200 cases have been treated with video-assisted esophagectomy in our hospital since 2013. Sometimes, postoperative chylothorax was observed. If the prophylactic ligation of thoracic duct should be advocated during esophagectomy? As far as we are concerned, both the benefits and adverse effects of thoracic duct ligation should be focused. On one hand, we admitted the fact that the incidence of postoperative chylothorax could be reduced with the ligation of the thoracic duct, thus, if the thoracic duct was suspected to be injured, the ligation should be performed without hesitation. On the other hand, the disadvantage of prophylactic ligation of the thoracic duct should be also considered. Some animal experiments showed that the ligation of thoracic duct leaded to decreased peripheral blood lymphocytes.[18] How about human being?.

The design of this project has initiated in 2014. After examination, a total of 60 patients were enrolled in this study. All patients were diagnosed as squamous cell carcinoma with endoscopic biopsy. The preoperative clinical stage of all patients was in T1N0M0 stage. Combined thoracoscopic-laparoscopic esophagectomy was performed by one group of surgeons. Vein blood was drawn before and after operation to detect lymphocyte and T lymphocyte subsets. After evaluating clinical data of all participants, no significant difference was observed between the two groups in age, sex, and stage. For both the ligation group and the nonligation group, the percentage of lymphocytes in WBCs and lymphocyte count were lower after operation. The percentage of T lymphocytes and CD4+ T lymphocytes was also significantly lower after operation (P < 0.05). The result was consistent with that of described in previous literature, and the reasons for the change in lymphocyte subsets may be the trauma of surgery and stress response in general anesthesia.[19]

Comparing the percentage of lymphocytes before operation, the mean value of ligation group was higher than that of in nonligation group (P > 0.05). The mean value of ligation group was lower than that of in the nonligation group after operation though P > 0.05. Comparing the results, we considered that the ligation of thoracic duct may lead to decreased percentage of lymphocytes. However, due to the insufficient number of samples, the difference was not significant.

Interestingly, we found the percentage of T lymphocytes and CD4+T lymphocytes of ligation group was significantly lower than that of in nonligation group after operation (P < 0.05). Lymphocytes and various types of immune cells in the blood circulation entered the postcapillary venule, then passed through high-walled endothelium of the postcapillary venues into the lymphatic tissue and lymphoid organs and thus entered the lymphatic circulation. Most of the lymph circulation was finally pooled into thoracic duct and then entered superior vena cava, thus returned into the blood circulation again. Due to the recirculation of lymphocytes, immune cells can communicate with each other, throughout the whole body and reasonably distribute in lymphatic tissues and organs.[20] In lymphoid organs or lymph tissue, many lymphocytes were participated in recycling, called lymphocyte recirculating pool, which was about ten times of the total number of lymphocytes in the blood. Therefore, as the most important main pipeline for draining lymphocytes, if the thoracic duct were ligated, peripheral blood lymphocytes would be bound to decrease. In general, recycling of T lymphocytes was more rapidly than that of B lymphocytes. Most of the B cells located in the bone marrow and aggregated in lymphatic follicles, but its number was much less than that of T lymphocytes in the thoracic duct.[20] Some experiments have indicated that memory CD4+T cells was the main force of lymphocyte in recycling, which could explain why the decrease of T lymphocytes, especially CD4+T cells, was more severe after ligation of thoracic duct.[21]

There were several communicating branches between thoracic duct and azygous vein, through which lymphocytes can enter the blood circulation after thoracic duct was ligated. During thoracoscopic esophagectomy, arch of azygos vein was generally ligated and transected for easer to free esophagus and clean lymph node, which make lymphocyte very hard to return to the heart through this way. It may be another reason that thoracic duct ligation during thoracoscopic esophagectomy made more effects on lymphocyte recycling than the traditional operations.

Comparing the postoperative chylothorax incidence between two group [Table 1], routine prophylactic thoracic duct ligation cannot significantly prevent postoperative chylothorax. Combined with the literature, we suggested that thoracic duct should not be prophylactically ligated in lower esophageal cancer in earlier stages, especially when there was no obvious sign of tumor invasion and thoracic duct injury. On the contrary, for the middle and upper segment esophageal carcinoma, especially tumor with serious invasion and mediastinal lymph node enlargement needing wide sweep, especially when thoracic duct was highly suspicious of injure, the prophylactic thoracic duct ligation is strongly recommended.

> Conclusion

Our study suggested that ligation of thoracic duct during video-assisted esophagectomy can lead to decrease of T lymphocyte and CD4+T lymphocyte, especially after arch of azygos vein has been transected. The thoracic duct should be selectively ligated during esophagectomy.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgment

First and foremost, I would like to show my deepest gratitude to my supervisor, Prof. Zhong-min Jiang doctoral tutor of Shandong University, chairman of the Department of Thoracic Surgery of Shandong Provincial Medical Association, who has provided with valuable guidance in every stage of the writing of this thesis. I would also like to thank all my colleagues for their encouragement and support. Last but not least, I would like to thank all the participants in the trial.